Endonuclease-resistant messenger rna and uses thereof

Abstract

The present disclosure provides messenger RNAs (mRNAs) with alterations which provide increased endonuclease resistance to the mRNA and methods and uses thereof.

1 . A method of increasing stability of an mRNA, comprising:
 (i) providing an mRNA comprising at least one endonuclease sensitive sequence motif, wherein the endonuclease sensitive sequence motif comprises the nucleotide sequence WGA, wherein W=adenine (A) or uracil (U); and   (ii) altering the at least one endonuclease sensitive sequence motif, thereby increasing stability of the mRNA.   
     
     
         2 . A method of increasing resistance and/or decreasing susceptibility of an mRNA to endonuclease activity, wherein the mRNA comprises at least one endonuclease sensitive sequence motif, wherein the endonuclease sensitive sequence motif comprises the nucleotide sequence WGA, wherein W=adenine (A) or uracil (U), the method comprising altering the at least one endonuclease sensitive sequence motif, thereby increasing resistance and/or decreasing susceptibility of the mRNA to endonuclease activity. 
     
     
         3 . A method of increasing the half-life of an mRNA, wherein the mRNA comprises at least one endonuclease sensitive sequence motif, wherein the endonuclease sensitive sequence motif comprises the nucleotide sequence WGA, wherein W=adenine (A) or uracil (U), the method comprising altering the at least one endonuclease sensitive sequence motif, thereby increasing the half-life of the mRNA. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the mRNA comprises a 5′ untranslated region (5′ UTR), an open reading frame (ORF) encoding a polypeptide, and a 3′ UTR, and wherein the 5′ UTR, ORF and/or 3′ UTR comprise at least one endonuclease sensitive sequence motif. 
     
     
         5 . The method of  claim 4 , wherein the 5′ UTR comprises the at least one endonuclease sensitive sequence motif. 
     
     
         6 . The method of  claim 4 , wherein the ORF comprises the at least one endonuclease sensitive sequence motif. 
     
     
         7 . The method of  claim 4 , wherein the 3′UTR comprises the at least one endonuclease sensitive sequence motif. 
     
     
         8 . The method of  claim 4 , wherein the 5′UTR and ORF each comprise at least one endonuclease sensitive sequence motif, and wherein the at least one endonuclease sensitive sequence motif is the same or is different. 
     
     
         9 . The method of  claim 4 , wherein the 5′UTR and 3′UTR each comprise at least one endonuclease sensitive sequence motif, and wherein the at least one endonuclease sensitive sequence motif is the same or is different. 
     
     
         10 . The method of  claim 4 , wherein the ORF and the 3′UTR each comprise at least one endonuclease sensitive sequence motif, and wherein the at least one endonuclease sensitive sequence motif is the same or is different. 
     
     
         11 . The method of  claim 4 , wherein the 5′UTR, ORF and 3′UTR each comprise at least one endonuclease sensitive sequence motif, and wherein the at least one endonuclease sensitive sequence motif is the same or is different. 
     
     
         12 . The method of any one of  claims 1 - 11 , wherein the endonuclease sensitive sequence motif is about 3-4, about 4-6, about 6-10, about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. 
     
     
         13 . The method of  claim 12 , wherein the endonuclease sensitive sequence motif is 3 nucleotides in length. 
     
     
         14 . The method of any one of  claims 1 - 13 , wherein altering the at least one endonuclease sensitive sequence motif comprises making a substitution or chemical modification of at least one nucleotide in the endonuclease sensitive sequence motif. 
     
     
         15 . The method of any one of  claims 1 - 13 , wherein altering the at least one endonuclease sensitive sequence motif comprises inserting one or more nucleotides into the motif, deleting one or more nucleotides from the motif, substituting one or more nucleotides in the motif, or a combination thereof. 
     
     
         16 . The method of any one of  claims 14 - 15 , wherein W=adenine (A), and wherein W is substituted with cytosine (C), guanine (G), or uracil (U). 
     
     
         17 . The method of any one of  claims 14 - 15 , wherein W=uracil (U), and wherein W is substituted with cytosine (C), guanine (G), or adenine (A). 
     
     
         18 . The method of any one of  claims 14 - 15 , wherein W=adenine (A), wherein W is preceded by an AG, and wherein AG is substituted with GC. 
     
     
         19 . The method of  claim 18 , wherein W is substituted with cytosine (C), guanine (G), or uracil (U). 
     
     
         20 . The method of any one of  claims 14 - 15 , wherein W=adenine (A), wherein W is substituted with guanine (G), wherein W is preceded by CG, and wherein the C is substituted with A. 
     
     
         21 . The method of any one of  claims 14 - 15 , wherein W=adenine (A), wherein W is substituted with cytosine (C) or uracil (U), wherein W is preceded by uracil (U) and cytosine (C), and wherein UC is substituted with AG. 
     
     
         22 . The method of any one of  claims 14 - 15 , wherein W=adenine (A), wherein W is substituted with cytosine (C), guanine (G), or uracil (U), wherein W is preceded by a UU, and wherein the first U comprising the UU is substituted with C. 
     
     
         23 . The method of any one of  claims 14 - 15 , wherein W=uracil (U), wherein W is substituted with cytosine (C), guanine (G), or adenine (A), wherein W is preceded by an AG, and wherein the AG is substituted with UC. 
     
     
         24 . The method of any one of  claims 14 - 15 , wherein W=uracil (U), wherein W is substituted with adenine (A) or guanine (G), wherein W is preceded by a CU, and wherein the C is substituted with U. 
     
     
         25 . The method of any one of  claims 14 - 15 , wherein W=uracil (U), wherein W is substituted with cytosine (C), wherein W is preceded by a UC, and wherein the UC is substituted with AG. 
     
     
         26 . The method of any one of  claims 14 - 15 , wherein W=adenine (A), and wherein the adenine (A) following the guanine (G) of the sequence motif is substituted with guanine (G). 
     
     
         27 . The method of any one of  claims 4 ,  7 ,  9 ,  10 , and  11 , wherein the sequence motif comprises the stop codon UGA, wherein the G is substituted with A, thereby forming the stop codon UAA. 
     
     
         28 . The method of any one of  claims 4 ,  7 ,  9 ,  10 , and  11 , wherein the sequence motif comprises the stop codon UGA, wherein the GA is substituted with an AG, thereby forming the stop codon UAG. 
     
     
         29 . The method of any one of  claims 4 ,  6 ,  8 ,  10  and  11 , wherein the at least one endonuclease sensitive sequence motif comprises the nucleotide sequence AGA, wherein the endonuclease sensitive sequence motif is a codon comprising the ORF, wherein the codon encodes the amino acid arginine, and wherein altering the at least one sequence motif results in the formation of a degenerate codon encoding arginine, and wherein the codon and the degenerate codon are different. 
     
     
         30 . The method of  claim 29  wherein the degenerate codon is selected from the group consisting of: AGG, CGU, CGC, CGA, and CGG. 
     
     
         31 . The method of any one of  claims 4 ,  6 ,  8 ,  10  and  11  wherein the at least one endonuclease sensitive sequence motif comprises the nucleotide sequence AGA, wherein the 5′ A is the 3 rd  position (wobble position) of a codon comprising the ORF, wherein the codon encodes an amino acid selected from the group consisting of: lysine, threonine, arginine, isoleucine, glutamine, proline, leucine, glutamic acid, alanine, glycine, valine, and serine; wherein altering the at least one endonuclease sensitive sequence motif results in the formation of a degenerate codon encoding the amino acid, wherein the codon and the degenerate codon are different. 
     
     
         32 . The method of any one of  claims 4 ,  6 ,  8 ,  10  and  11  wherein the at least one endonuclease sensitive sequence motif comprises the nucleotide sequence UGA, wherein the U is the 3 rd  position (wobble position) of a codon comprising the ORF, wherein the codon encodes an amino acid selected from the group consisting of: asparagine, threonine, serine, isoleucine, histidine, proline arginine, leucine, aspartic acid, alanine, glycine, valine, tyrosine, cysteine, and phenylalanine, wherein altering the at least one endonuclease sensitive sequence motif results in the formation of a degenerate codon encoding the amino acid, and wherein the codon and the degenerate codon are different. 
     
     
         33 . The method of any one of  claims 4 ,  6 ,  8 ,  10 , and  11 , wherein the at least one endonuclease sensitive sequence motif comprises the nucleotide sequence AGA, wherein the G is the 3 rd  position (wobble position) of a codon comprising the ORF, wherein the codon encodes an amino acid selected from the group consisting of: lysine, glutamine, and glutamic acid, wherein altering the at least one endonuclease sensitive sequence motif results in the formation of degenerate codon encoding the amino acid, and wherein the codon and the degenerate codon are different. 
     
     
         34 . The method of any one of  claims 4 ,  6 ,  8 ,  10 , and  11 , wherein the at least one endonuclease sensitive sequence motif comprises the nucleotide sequence UGA, wherein the G is the 3 rd  position (wobble position) of a codon comprising the ORF, wherein the codon encodes an amino acid selected from the group consisting of: leucine and valine, wherein altering the at least one endonuclease sensitive sequence motif results in the formation of a degenerate codon encoding the amino acid, and wherein the codon and the degenerate codon are different. 
     
     
         35 . The method of any one of  claims 1 - 34 , wherein altering the at least one endonuclease sensitive sequence motif increases stability of the mRNA. 
     
     
         36 . The method of any one of  claims 1 - 35 , wherein altering the at least one endonuclease sensitive sequence motif increases the half-life of the mRNA. 
     
     
         37 . The method of any one of  claims 2  and  4 - 36 , wherein increased resistance of the mRNA to endonuclease activity is determined relative to an unaltered mRNA, wherein the unaltered mRNA contains at least one additional endonuclease sensitive sequence motif relative to the altered mRNA. 
     
     
         38 . An mRNA produced by any one of the preceding methods. 
     
     
         39 . An mRNA comprising:
 (i) a 5′ UTR;   (ii) an ORF encoding a polypeptide; and   (iii) a 3′ UTR, wherein the 3′ UTR comprises the nucleotide sequence of SEQ ID NO: 1, comprising from 5′ to 3′ a first endonuclease sensitive sequence motif (UGA1), a second endonuclease sensitive sequence motif (UGA2), and a third endonuclease sensitive sequence motif (UGA3), wherein at least one of UGA1, UGA2 and/or UGA3 is altered by deletion, substitution or insertion.   
     
     
         40 . The mRNA of  claim 39 , wherein UGA1 is altered by deletion. 
     
     
         41 . The mRNA of  claim 39  or  40 , wherein UGA2 is altered by a substitution. 
     
     
         42 . The mRNA of any one of  claims 39 - 41 , wherein UGA3 is altered by a substitution. 
     
     
         43 . The mRNA of any one of  claims 39 - 40 , wherein UGA2 is altered by substitution of G with cytosine (C) or adenine (A). 
     
     
         44 . The mRNA of  claim 43 , wherein UGA2 is altered by substitution of G with cytosine (C). 
     
     
         45 . The mRNA of any one of  claims 39 - 40 , wherein UGA3 is altered by substitution of G with cytosine (C), or adenine (A). 
     
     
         46 . The mRNA of  claim 45 , wherein UGA3 is altered by substitution of G with cytosine (C). 
     
     
         47 . The mRNA of any one of  claims 39 - 40 , wherein UGA2 and UGA3 are altered by substitution of G with cytosine (C), or adenine (A). 
     
     
         48 . The mRNA of  claim 47 , wherein UGA2 and UGA3 are altered by substitution of G with cytosine (C). 
     
     
         49 . An mRNA comprising:
 (i) a 5′ UTR;   (ii) an ORF encoding a polypeptide; and   (iii) a 3′ UTR comprising the nucleotide sequence of SEQ ID NO: 2.   
     
     
         50 . The mRNA of any one of  claims 39 - 49 , wherein the ORF encodes a polypeptide of interest. 
     
     
         51 . The mRNA of  claim 50 , wherein the polypeptide of interest is a therapeutic polypeptide. 
     
     
         52 . The mRNA of any of  claims 39 - 51 , wherein the 3′ UTR comprises a poly-A region. 
     
     
         53 . The mRNA of  claim 52 , wherein the 3′ UTR further comprises a terminal 3′-stabilizing region comprising 1 to 500 nucleosides. 
     
     
         54 . The mRNA of  claim 53 , wherein the 3′-stabilizing region comprises a plurality of alternative nucleosides. 
     
     
         55 . The mRNA of  claim 54 , wherein the alternative nucleoside is L-adenosine 
     
     
         56 . The mRNA of any one of  claims 53 - 55 , wherein the 3′-stabilizing region is conjugated to the 3′ UTR by a linker. 
     
     
         57 . A lipid nanoparticle comprising the mRNA of any one of  claims 38 - 56 . 
     
     
         58 . A pharmaceutical composition comprising the mRNA of any one of  claims 38 - 56 , or the lipid nanoparticle of  claim 57 , and a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         59 . The lipid nanoparticle of  claim 57 , and an optional pharmaceutically acceptable carrier, diluent, or excipient, or the pharmaceutical composition of  claim 58 , for use in treating or delaying progression of a disease or disorder in a subject, wherein the treating or delaying progression of the disease or disorder comprises administration of the lipid nanoparticle, and an optional pharmaceutically acceptable carrier, diluent, or excipient, or the pharmaceutical composition. 
     
     
         60 . Use of a lipid nanoparticle of  claim 57 , and an optional pharmaceutically acceptable carrier, or the pharmaceutical composition of  claim 58 , in the manufacture of a medicament for treating or delaying progression of a disease or disorder in a subject, wherein the medicament comprises the lipid nanoparticle, and an optional pharmaceutically acceptable carrier, or the pharmaceutical composition and wherein the treatment comprises administration of the medicament. 
     
     
         61 . A kit comprising a container comprising the mRNA of any one of  claims 38 - 56 , and an optional pharmaceutically acceptable carrier, the lipid nanoparticle of  claim 57 , and an optional pharmaceutically acceptable carrier, or the pharmaceutical composition of  claim 58 , and a package insert comprising instructions for administration of the mRNA, the lipid nanoparticle, or the pharmaceutical composition for treating or delaying progression of a disease or disorder in a subject. 
     
     
         62 . A method of treating or delaying progression of a disease or disorder in a subject in need thereof, the method comprising administering the mRNA according to any one of  claims 38 - 56 , the pharmaceutical composition according to  claim 58 , or the lipid nanoparticle according to  claim 57 , thereby treating or delaying progression of the disease or disorder in the subject.